Tissue Engineering of Cartilage
Dissertation to obtain the Degree of Doctor of Natural Sciences (Dr. rer. nat.)
from the Faculty of Chemistry and Pharmacy University of Regensburg
Presented by
Daniela Eyrich
from Bad Kissingen
April 2006
Pharmaceutical Technology of the University of Regensburg.
The thesis was prepared under supervision of Prof. Dr. Achim Göpferich.
Submission of the PhD. application : 24.04.2006
Date of examination : 18.05.2006
Examination board : Chairman: Prof. Dr. Elz 1. Expert: Prof. Dr. Göpferich 2. Expert: PD Dr. Staudenmaier 3. Examiner: Prof. Dr. Heilmann
‘If we consult the standard surgical writers from Hippocrates down to present age, we shall find that an ulcerated cartilage is found to be a very troublesome disease [...] and that when destroyed, it is never recovered.’
W. Hunter
Roy Soc London, Phil Trans 9:267-271 (1743)
Table of Contents
Chapter 1 Goals of the Thesis ...7
Chapter 2 Introduction - Fibrin in Tissue Engineering ...11
Chapter 3 Factors Influencing Chondrocyte Behavior and Development of Cartilaginous Tissue in Three-Dimensional Fibrin Gel...35
Chapter 4 Long-Term Stable Fibrin Gels for Cartilage Engineering...63
Chapter 5 Proliferation on a Fibrin Surface Enhances the Potential of Expanded Chondrocytes to Generate Engineered Cartilage ...91
Chapter 6 Cartilage Tissue Engineering Using Human Chondrocytes in a Fibrin Matrix...111
Chapter 7 Combination of Long-Term Stable Fibrin Gels and Polymeric Scaffolds for Cartilage Tissue Engineering ...135
Chapter 8 In Vitro and in Vivo Cartilage Engineering Using a Combination of Long-Term Stable Fibrin Gels and Polycaprolactone-Based Scaffolds ...163
Chapter 9 Summary and Conclusions ...189
Appendices Abbreviations...197
Curriculum Vitae ...199
List of Publications ...201
Acknowledgment ...205
Chapter 1
Goals of the Thesis
Since the beginning of the 1990s a plethora of research approaches towards the engineering of cartilage have been undertaken. However, a general standard method for generation of cartilage tissue equivalent and their clinical application is still lacking. The goal of this thesis is based on the project ‘Bavarian Research Cooperation (‘Bayerischer Forschungsverbund’) for Tissue Engineering and Rapid Prototyping’ (ForTEPro), which is a multi-partner network of research groups from university, hospital, and industry supported by a grant from the Bavarian Research Foundation (‘Bayerische Forschungsstiftung’) in the years 2002 to 2005. The major goal of the overall project was the development of individually customized implants for anatomical cartilage and bone defects of the head and the musculo-skeletal system. A subgroup of the research consortium aimed at the generation of cartilage for facial plastic and reconstructive surgery, especially external ear. In principle, autologous chondrocytes were to be isolated, suspended in a hydrogel, and the gel was subsequently to be injected into a polymeric scaffold that was individually shaped using newly established rapid prototyping technologies.
The overall goal of the thesis was, within the ForTEPro project, the utilization of fibrin for tissue engineering of cartilage. The hydrogel fibrin is a well-investigated medical device and has been used for over 20 years in clinical practice and surgery (chapter 2). However, commonly employed commercially available preparation kits often result in gels that are unstable in cell culture, shrink, and dissolve within a few weeks, which makes them unsuitable for many applications in shape-specific tissue engineering [1, 2]. Therefore, as a first major step, fibrin glue parameters were determined influencing appearance and stability of the gel in order to obtain a long-term shape stable scaffold material for culture of cells. Subsequently, these optimized fibrin gels were demonstrated to be suitable for the use in cartilage tissue engineering. Therefore, primary bovine chondrocytes were suspended in the gel system and in detail analyzed regarding cell morphology, cell proliferation as well as extracellular matrix production and distribution to establish a chondrocyte-fibrin culture system. In particular, the influence of fibrinogen concentration, cell density, and different culture conditions on formation of cartilaginous tissue was investigated. With the objective to generate a coherent cartilaginous tissue using primary bovine chondrocytes, the minimum initial cell number required for the formation of an adequate uniform extracellular matrix was determined and the effect of exogenous insulin was examined (chapter 3 and 4).
Chondrocytes in monolayer rapidly dedifferentiate from a cartilaginous phenotype towards a more fibroblast-like phenotype accompanied by production of inadequate extracellular matrix molecules. Therefore, as a next step within this thesis, the long-term stable fibrin gels were tested for the use in the proliferation of primary bovine chondrocytes, with specific regard to its potential to retain the ability of the thus expanded chondrocytes to form engineered cartilage (chapter 5). Cells that migrated out of fibrin and proliferated on the gel were re-seeded into three-dimensional fibrin gels in order to evaluate differentiation capacity compared to cells proliferated on conventional cell culture surface.
The main goal of chapter 6 was to transfer the established fibrin culture method from using bovine cells to human cells, with regard to a future clinical application. Human chondrocytes were isolated from small nasal and articular biopsies, and seeded into the established fibrin gels. Based on previous studies in another cartilage engineering culture system [3, 4], a special focus was set on the effect of insulin and insulin-like growth factor-I (IGF-I) to enhance formation of new human cartilaginous tissue.
As an important step within the ForTEPro consortium, the fibrin gels were combined with polymeric scaffolds (chapter 7). Hydrogels generally enable a good cell distribution, but often lack adequate mechanical strength [5]. Highly porous solid scaffolds can provide sufficient load-bearing capacity, however, many scaffold systems lack an adequate cell seeding efficiency, cell distribution and a subsequent sufficient extracellular matrix development [6]. In order to overcome the disadvantages associated with either system, bovine chondrocytes were suspended in the optimized fibrin gel and subsequently injected into newly developed polycaprolactone-based scaffolds, based on results from research partners. Additionally, the results were compared with injecting the cell-fibrin suspension into commonly used PGA meshes as well as PLGA scaffolds. With regard to the overall goal of the generation of a prototype of an external ear within ForTEPro, the cell-fibrin suspension was injected into a polycaprolactone-based scaffold in the shape of the cartilage part of an external human ear, and maintenance of ear shape as well as new tissue formation within the scaffold was evaluated.
In a follow-up study, the established composite constructs consisting of bovine chondrocytes suspended in fibrin gel and distributed within a polycaprolactone-based scaffold was further tested in vivo (chapter 8). The constructs were implanted into the back of nude mice and examined regarding formation of new cartilaginous tissue after 1, 3, and 6 months. In particular, tissue development was compared to constructs prepared with cells seeded into fibrin gel alone as well as cells seeded directly onto the polymeric
scaffold. Furthermore, a special focus was set on the effect of in vitro pre-cultivation of the constructs prior to implantation on subsequent in vivo development of the cartilaginous tissue.
References
[1] Meinhart J, Fussenegger M, and Hobling W, 'Stabilization of fibrin-chondrocyte constructs for cartilage reconstruction', Ann Plast Surg 42 (6), 673-678 (1999).
[2] Fussenegger M, Meinhart J, Hobling W, Kullich W, Funk S, and Bernatzky G, 'Stabilized autologous fibrin-chondrocyte constructs for cartilage repair in vivo', Ann Plast Surg 51 (5), 493-498 (2003).
[3] Kellner K, Schulz M B, Goepferich A, and Blunk T, 'Insulin in tissue engineering of cartilage: a potential model system for growth factor application', J Drug Target 9 (6), 439-448 (2001).
[4] Blunk T, Sieminski A L, Gooch K J, Courter D L, Hollander A P, Nahir A M, Langer R, Vunjak-Novakovic G, and Freed L E, 'Differential effects of growth factors on tissue-engineered cartilage', Tissue Eng 8 (1), 73-84 (2002).
[5] Drury J L and Mooney D J, 'Hydrogels for tissue engineering: Scaffold design variables and applications', Biomaterials 24 (24), 4337-4351 (2003).
[6] Grad S, Kupcsik L, Gorna K, Gogolewski S, and Alini M, 'The use of biodegradable polyurethane scaffolds for cartilage tissue engineering: Potential and limitations', Biomaterials 24 (28), 5163-5171 (2003).
Chapter 2
Fibrin in Tissue Engineering
Daniela Eyrich 1, Achim Goepferich 1, Torsten Blunk 1
1 Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93051 Regensburg, Germany
in ‘Tissue Engineering’, Adv Exp Med Biol, Fisher J P (ed.), 585 (2006), in press as review
Introduction
Every year, millions of people suffer loss or failure of tissue or organs due to an accident or clinical condition. A revolutionary strategy to treat these patients is the engineering of the lacking organ or tissue with autologous cells in combination with polymeric matrices [1].
For several years there has been enormous interest in hydrogels as a soft scaffold for tissue engineering [2-4]. In nearly every intact native tissue, cells are held within an extracellular matrix that modulates tissue development, homeostasis and regeneration. Hydrogels are structurally similar to the extracellular matrix of many tissues and are considered to be biocompatible. They have many potential functions in the field of tissue engineering and, thus, must fulfill many different requirements to promote new tissue growth, depending on their application. Hydrogels act as a space filling agent and three-dimensional structure to organize the expanded cells, to maintain a specific shape and structural integrity, and to direct growth and formation for adequate new tissue development. In general, hydrogels can be processed under relatively mild conditions suitable for many cell types.
Furthermore, the liquid components of hydrogels may be easily delivered into the patient’s defect in a minimally invasive manner.
Factor XIII
Factor XIIIa
Ca2+
Fibrinogen
Fibrin soluble monomers
Fibrin unstable
clot
Fibrin stable clot Ca2+
Thrombin
Factor XIII
Factor XIIIa
Ca2+
Fibrinogen
Fibrin soluble monomers
Fibrin unstable
clot
Fibrin stable clot Ca2+
Thrombin
Factor XIII
Factor XIIIa
Ca2+
Fibrinogen
Fibrin soluble monomers
Fibrin unstable
clot
Fibrin stable clot Ca2+
Thrombin
Fig. 1: Scheme of fibrin gel formation.
One of the most widely used hydrogels is fibrin. Fibrin glue is a commonly used surgical haemostatic agent and has been commercially available for over 20 years in surgery and clinical practice. The hydrogel fibrin is a polypeptide consisting of the plasma components fibrinogen and thrombin. Physiologically, fibrin formation occurs as the final step in the natural blood coagulation cascade, producing a clot that assists wound healing. After activation with calcium ions, thrombin cleaves small peptides from the fibrinogen chain to produce soluble fibrin monomers [5]. These monomers are covalently cross-linked through the action of factor XIII to form an insoluble, polymerized fibrin clot [6] (Fig. 1). In recent years, fibrin has been utilized for different applications in the field of tissue engineering with specific physical and biological requirements. The current use of fibrin for each of these categories will be subsequently reviewed.
Fibrin glue in surgery and clinical practice
Several reviews published in recent years have focused on uses of fibrin glue, also referred to as fibrin tissue adhesive or fibrin sealant, in clinical and surgical practice [7-11]. In the literature, fibrin was first mentioned more than 90 years ago. It has been documented that fibrinogen combined with thrombin was used to improve the adhesion of skin grafts of soldiers with burn injuries during the Second World War [7].
A commercial product has been available in Europe and Japan since the 80s, whereas fibrin glue was not FDA approved until 1998 because of possible viral contamination. At the moment, fibrin sealant is considered the most effective physiological tissue adhesive available.
There are a number of commercially available fibrin products with different amounts and origins of the components [7, 9]. The concentration of fibrinogen, varying between 40 and 125 mg/ml, is directly correlated to the tensile strength of the fibrin clot, whereas the concentration of thrombin influences the degree and speed of clotting. The latter proves useful for quick haemostasis to prevent blood loss (e.g., in suturing of vessels) or in surgical procedures involving careful glue adjustment to fit a tissue or organ [9]. Within 3 days of application, a preliminary granulation tissue with a large number of wound healing cells is present and is subsequently replaced with collagen fibers one to two weeks later.
During normal wound healing the fibrin glue is absorbed within days to weeks depending on the type of sealant and location of application [12]. The majority of glues contain an anti-fibrinolytic component to reduce the degradation rate. A common agent is the protease
inhibitor aprotinin, which inhibits human trypsin and plasmin by blocking the active sites of the enzymes. Due to the fact that fibrin is a physiological blood component, it is considered to be biocompatible and biodegradable. However, despite a number of rigorous national and international guidelines during manufacturing to assure the high quality of commercial fibrin components, the risk of viral infection or foreign body response, especially due to bovine components, still exists [7]. Additionally, the development of antibodies against coagulation plasma proteins has been documented after application of bovine thrombin resulting in significant anticoagulation [10]. Although most of the commercial products now contain human fibrinogen and thrombin, the majority still contain bovine aprotinin, which was shown to cause hypersensitivity, especially after repeated administration [10]. Autologous preparation methods have been described [13-16]
to prevent these foreign body reactions, however, the composition, resulting appearance, and mechanical strength of these gels depend on the patient’s physique and constitution.
Therefore, there is a great interest in optimizing these methods leading to standardization and validation in clinical practice.
The most prevalent application of fibrin in clinical practice is its use as a haemostatic agent, especially in heparinized or coagulopathic patients, to reduce operative bleeding, e.g., in cardiovascular surgery. The application is most effective when polymerized prior to the onset of bleeding, for example in surgery of a vascular anastomosis [8]. When using fibrin sealants or sprays as adjunct to sutures, a better wound healing and optimal wound integrity results in operative locations where the use of conventional sutures is not feasible or would result in intense bleedings [8, 10].
Fibrin polymers play a key role in tissue and organ sealing, particularly in plastic and reconstructive surgery, including skin grafting [8]. Exact adjustment is possible, bleeding is reduced, fewer sutures are necessary, the length of the operation time is shortened, and fewer post-operative infections occur.
Fibrin glue applications are common in other important fields of clinical practice, including thoracic, orthopedic, neuro-, and oral surgery [7, 8, 12]. Since the recent approval of fibrin glues by the FDA, the number of clinical applications has increased dramatically and companies have started to investigate and improve the use of the sealant in more diverse surgical settings.
Principles and methods of fibrin application in tissue engineering
In order for fibrin gels to be utilized as a tissue scaffold, the material must provide an environment enabling adequate cellular function, e.g., cell migration, proliferation, and differentiation, and must allow for tissue development. For example, it has been shown that chondrocytes in fibrin gel retain their round and vital morphology, do not dedifferentiate, and produce extracellular matrix [17, 18]. The glue components fibrinogen and thrombin are thought to modulate the attachment, migration, and proliferation of different cell types, e.g., chondrocytes, fibroblasts, smooth muscle cells, or keratinocytes [19-23]. Fibrinogen possesses a specific peptide chain, also referred to as a heptide, that contributes to cell attachment and binding primarily of mesenchymal cells, e.g., fibroblasts, endothelial cells, and smooth muscle cells [24]. This data is consistent with a study published by Brown, who investigated the effect of cross-linking various fibrin chains on fibroblast migration [25]. An important factor modulating fibroblast movement into a fibrin gel has been shown to be factor XIII which mediates the cross-linking of the fibrin α-chains [26].
The variation of fibrin parameters can generate gels with different mechanical stiffnesses.
These mechanical properties potentially influence the gene expression of different growth factors and cytokines, e.g., of human dermal fibroblasts [27]. Important parameters affecting fibrin characteristics are thrombin, fibrinogen, and calcium concentration, ionic strength, and pH, resulting in either more rigid and stable or more soft and soluble gels [28-30], although the exact contributions of each parameter are still not fully understood.
Nevertheless, it may be possible to tailor specific structural fibrin features for specific cell types and for a particular application to modulate individual cell proliferation, migration and differentiation [31].
Another fibrin glue characteristic is an increasing instability and solubility over time, due to fibrinolysis [28]. Commercially available fibrin sealants tend to shrink and disintegrate in vitro and in vivo after a few days and almost completely dissolve within 4 weeks [17, 32-35]. While this could be an advantage in wound sealing or other surgical applications in which dissolution is desired after closure of the defect [7, 9, 12], this can represent a major problem for use as a shape-specific scaffold in tissue engineering. Here, long-term stability is necessary to provide enough time for cell proliferation and matrix production. Therefore, fibrinolysis inhibitors, mostly protease inhibitors, e.g., aprotinin, ε-amino caproic acid or
tranexamic acid, are used within the fibrin gel and/or as a supplement to the cell culture medium; they can help slow down degradation and, thus, stabilize the fibrin gel shape [32, 36, 37]. As a result, degradation of the temporary matrix may be controlled for specific purposes.
The application of fibrin in tissue engineering can be grouped into three main areas: fibrin as cell matrix material alone, fibrin as a cell matrix material combined with a polymeric scaffold, and as delivery system for growth factors or other therapeutic agents.
A simple method for the use of fibrin as a scaffold material involves suspending primary or expanded cells in a liquid component of the fibrin glue with subsequent polymerization in suitable cell culture plates. The resulting three-dimensional construct may be cultivated in vitro to obtain an adequate tissue for re-implantation. In addition to the application as scaffold in vitro, the fibrin system can also be used as cell delivery vehicle in vivo. Cells suspended in fibrin glue can be directly injected into a defect in a minimally invasive procedure with little stress for the patient; the fibrin gel can be polymerized in vivo in the desired three-dimensional shape, at the same time ensuring the retention of the cells at the injection site [22, 36].
An alternative strategy for tissue engineering is the combination of hydrogels with polymeric scaffolds. Highly porous solid scaffolds can provide sufficient load-bearing capacity for the process of implantation and for structural integrity in vivo. However, many scaffold systems lack adequate cell seeding efficiency, sufficient cell distribution and subsequent sufficient extracellular matrix synthesis and deposition. In contrast, fibrin gels generally incorporate all of the applied cells and enable a good cell distribution providing the requirements for a coherent tissue development, but often lack adequate biomechanical strength and volume stability [34, 38-40]. Therefore, the advantages of fibrin glue combined with favorable characteristics of synthetic or naturally derived polymeric scaffolds can be utilized to develop a simple, stable composite for implantation. This way tissue development in the desired three-dimensional shape can be achieved; furthermore the time for tissue development may be reduced as compared to the use of either system alone [41]. This strategy has been successfully applied in tissue engineering of urothelium [42], cartilage [43-45], and cardiovascular engineering [41].
Fibrin can also be applied as delivery system for the release of growth factors, cytokines or other bioactive molecules to control cell adhesion, proliferation, migration, differentiation, and matrix production. Many growth factors bind to fibrin, bFGF and VEGF are even supposed to bind specifically. Alternatively, such proteins can be incorporated into the gel
during polymerization [46-48]. Fibrin is known to protect growth factors against denaturation and proteolysis in vitro and in vivo [49]. Furthermore, when applied in vivo the presence of the growth factor in the defect is maintained over a long time. Additionally, the kinetics of growth factor release can be controlled by varying fibrinogen and thrombin concentration as well as by the addition of degradation inhibitors. Factors released from fibrin gels used for tissue regeneration include bFGF, VEGF, NTF, ECGF, GDNF and NGF [48, 50-62]. Hubbell et al. developed an innovative technology for growth factor delivery on the basis of a combination of fibrin and heparin, utilizing the ability of heparin to stabilize the bioactivity of growth factors and control their release [49]. A fibrin gel was modified by covalently binding exogenous bi-domain peptides with a heparin-binding domain using the transglutaminase activity of factor XIIIa during coagulation. These peptides can bind heparin and subsequently heparin-binding growth factors (Fig. 2). This approach was successfully applied in the controlled release of different growth factors [53, 54, 62-67] either by passive release or facilitated by enzymatic factors secreted by migrating cells.
Fig. 2: Fibrin matrix as gel for delivery system utilizing the growth factor binding properties of heparin. A bi-domain peptide, containing a factor XIIIa substrate and a heparin-binding domain, is covalently cross-linked to the fibrin matrix during coagulation.
Heparin is immobilized to the heparin-binding domain of the peptide by electrostatic interactions. Heparin-binding growth factors are immobilized by binding to the immobilized heparin within the fibrin matrix (with permission of Elsevier; Ref: 62).
Andree et al. established a method to deliver EGF expression plasmids from a fibrin matrix to a human keratinocyte culture system. These plasmids can enhance keratinocyte proliferation during expansion in vitro as well as directly after transplantation of the cells in combination with a fibrin matrix into a skin defect in vivo [68].
Besides release of growth factors, the local delivery of antibiotics and chemotherapeutic agents from a fibrin gel could be beneficial in clinical applications [69, 70]. Skin replacement therapies, a common application in tissue engineering, carries a high risk of infection during the implantation procedure that could be moderated by the release of antibiotics from the tissue replacement itself. Unfortunately, the time span of release from fibrin hydrogels is rather short due to the fast diffusion of the small molecules. Release may be prolonged, however, by varying drug concentration or by use of fibrin insoluble antibiotics [71]; these drugs dissolve slowly inside the defect due to low hydrophilicity.
Fibrin in the engineering of specific tissues
Skin tissue
Numerous authors have investigated fibrin glue for skin tissue engineering during the last years. Not only can fibrin be used as a graft sealant as well as a haemostatic and antibacterial agent [72], fibrin is a functional scaffold material for engineering of skin tissue. It has been shown that fibrinogen stimulates the migration of epidermal cells and keratinocytes [73-75]. Fibronectin, a specific glycoprotein in fibrin glue, enhances cellular migration during wound healing [76, 77]. A common clinical application consists of a single cell suspension of in vitro expanded autologous human keratinocytes in fibrin sealant and delivery directly into the skin defect. The cell-glue system adheres and spreads over the defect resulting in re-epithelialization within a few weeks [76, 78, 79].
Additionally, cultivation of keratinocytes onto a fibrin layer in vitro maintains the status of differentiation [80].
An alternative therapy for chronic wounds and severe burns includes isolation of a small biopsy of the patient’s skin, expansion of the resultant single keratinocytes in vitro onto a supportive 3T3 feeder layer and transfer of the developed epidermal sheet directly to the wound. However, this is an expensive and time-consuming process and careful enzymatic detachment and handling of the cell sheet is critical. The application of fibrin as culture bed during expansion and transfer of the cell-fibrin construct facilitates and accelerates the
operative procedure [81]. Meana et al. investigated a fibrin gel either with or without human fibroblasts as a base for a dermal equivalent. Only in the group with fibroblasts, cultivation of even of low cell numbers of primary keratinocytes on this gel system resulted in a newly developed epithelium within 10-14 days (Fig. 3). The cell layer was anually detached from the culture flask without enzymatic treatment and could be easily transplanted into the skin defect [82]. Gorodetsky et al. developed an innovative technology to deliver cells from fibrin-derived microbeads instead of conventional fibrin gel systems [83]. These biodegradable microbeads, 50-200µm in diameter, represent a simple provisional matrix and cell carrier with good attachment properties for different cell types. Fibroblasts seeded at low density can proliferate in vitro on the fibrin particles and may be easily transferred from the culture plate into specific defects for wound healing.
The cell-seeded microbeads showed improved formation of granulation tissue in pig wound healing as compared to fibrin-derived microbeads alone. This strategy may be transferable to the engineering of other tissues as well.
Fig. 3: Fibrin as an approach to generate a dermal equivalent: keratinocytes were cultured on a fibroblast-containing fibrin gel. Histological appearance after 15 days of culture (H&E staining, x 250) (with permission of Elsevier; Ref: 82).
Vascular tissue
There is a tremendous demand for blood vessel repair, especially in cardiovascular surgery due to the high number of patients with arteriosclerosis. The investigation of fibrin gel parameters and the effects of exogenous factors added to fibrin scaffolds on inducing cells
to form vascular tissue within these gels are important first steps towards a clinical solution [84, 85]. Koolwijk et al. used in vitro experiments to analyze the effect of TNFα, bFGF and VEGF on human microvascular endothelial cells seeded on fibrin gels to form capillary-like tubular structures for angiogenesis [84]. The data showed that the inflammatory mediator TNFα is necessary in addition to bFGF and/or VEGF for development of capillary-like structures by endothelial cells.
Moreover, fibrin gels can serve as a three-dimensional matrix molded to the exact structure of vessels. Jockenhoevel et al. developed a method towards the engineering of valve conduits [86]. Fibroblasts were suspended in a fibrin gel that was polymerized in silicone- coated aluminum molds. 2 mm thick constructs were cultivated in vitro and cells subsequently produced collagen bundles, an element of valve conduits. In another study, the same group investigated methods to prevent the shrinkage of fibrin gels so they could be used in cardiovascular engineering. In addition to supplementing protease inhibitors to the culture medium, the mechanical and chemical fixation of gels onto culture plates was tested [40]. Matrix analysis and histology showed the best collagen synthesis and tissue development using a chemical border fixation onto culture plates. In contrast, Mol et al.
discussed the possible advantage of shrinking fibrin gels for vascular engineering leading to higher mechanical forces inside the construct and thereby potentially enhancing collagen production [41].
Furthermore, fibrin gels were tested as a scaffold for cardiovascular tissue engineering using myofibroblasts. Ye et al. suspended human myofibroblasts in a fibrin matrix [39].
Microscopy showed homogenous cell growth and collagen synthesis. Additionally, a higher concentration of aprotinin supplemented to the medium resulted in improved gel stability and enhanced tissue development. Cummings et al. investigated the morphological and mechanical properties of fibrin gel, collagen type I gel, and a combination of both for vascular engineering using rat aortic smooth muscle cells. A combination of rigid, less elastic collagen with a weaker, more instable fibrin gel resulted in higher ultimate tensile stress, increased toughness, and increased gel compaction compared to each system alone [87]. As an alternative, the variation in fibrin parameters resulting in more stable and rigid gels may be a means to avoid the more complex combinations with another gel system for this kind of application.
Bone tissue
Another potential field of application of fibrin is the healing of critical size defects of bone, although there are only a few papers published on the topic so far. One strategy, again, is the delivery of cells suspended in a fibrin gel directly into the bone defect. Isogai et al.
injected a periosteal cell-fibrin mixture into the dorsum of athymic mice. Histology showed new bone development and western blot assay demonstrated production of osteopontin, a specific protein in bone tissue [88]. Ng et al. investigated the potential of suspending cells derived from four different sites of the body in fibrin glue for three- dimensional bone constructs in vivo [89]. Osteoprogenitor cells isolated from periosteum showed best results, whereas using cells derived from cancellous and cortical bone as well as bone marrow resulted in less bone-forming activity.
Bensaid et al. tested the potential of fibrin as a scaffolding structure for mesenchmal stem cells in bone tissue engineering in vitro [22]. They varied fibrinogen concentration and thrombin activity and analysed the effect on cell spreading and proliferation in vitro. Perka et al. compared the cultivation of periosteal cells on PLGA polymer fleece and on fibrin beads. Both groups were cultivated in vitro for 14 days and subsequently implanted into metadiaphyseal ulna defects of white rabbits [90]. Histological and radiological analysis showed intense bone formation in both groups.
Karp et al. combined fibrin glues containing different thrombin concentrations with interconnecting, macroporous PLGA scaffolds for use in bone engineering [91]. However, no difference was seen between the control group (only PLGA scaffold, no fibrin) and the group with low thrombin concentration, whereas scaffolds filled with fibrin gel containing high thrombin concentrations showed less bone formation. Fibrin parameters may be optimized for this kind of application to obtain improved cell migration and matrix production.
Finally, Haisch et al. investigated a method to induce transdifferentiation of articular chondrocytes into bone-forming cells with addition of corticosteroids [92]. Auricular rabbit chondrocytes were suspended in fibrin or agarose and the mixture was injected into polymer fleeces. Constructs were subsequently implanted subcutaneously into the ridge of New Zealand rabbits with and without methylprednisolone treatment. Histology showed that the simple injection of corticosteroids prevented fibrous tissue formation and enhanced bone development.
Cartilage tissue
Fibrin is widely used in approaches to tissue engineering of cartilage. Fortier et al. tested fibrin as a matrix for engineering articular cartilage in vitro using equine chondrocytes.
Positive effects of exogenously applied IGF-I and TGF-β on chondrocyte matrix synthesis was shown [93, 94]. In another study, autologous fibrinogen was demonstrated to better maintain differentiation of chondrocytes compared to commercially available fibrinogen [18]. To further enhance cartilage development, Hunter et al. tested the influence of oscillatory compression on development of chondrocyte-fibrin constructs [95]. Though dynamic compression has frequently been shown to stimulate matrix production and gene expression in tissue, mechanical stimulus of the fibrin constructs resulted in the inhibition of cartilage matrix production. The effect of mechanical stimulation may depend on the structure and mechanical properties of the fibrin gel itself, which in turn depends on the concentration of the individual components and has to be tested for specific applications.
Several studies were published showing tissue engineering of cartilage after subcutaneous implantation in mice. Silverman et al. suspended swine chondrocytes in fibrin gel, injected the cell-fibrin mixture directly into the subcutaneous back of nude mice and determined the optimal fibrinogen and cell concentration required for adequate tissue development [38].
Sims et al. were the first who reported about the successful tissue engineering of cartilage in the back of nude mice after construct preparation in vitro [96]. Bovine chondrocytes were isolated and suspended in fibrinogen; after polymerization with bovine thrombin, the constructs were implanted subcutaneously into mice. Quantitative analysis and histology demonstrated that differentiated cells were producing cartilaginous extracellular matrix after 6 and 12 weeks. Using the same method, the growth and development of swine chondrocytes from different sites of the body as well as the volume stability of the constructs were explored [97]. Cultivation of articular chondrocytes resulted in decreased construct volume, whereas auricular chondrocytes produced high amounts of extracellular matrix resulting in construct overgrowth. These results indicate the importance of cell source.
Furthermore, the cultivation of chondrocytes in fibrin gels with expanded highly porous polytetrafluoroethylene as a stabilizing pseudoperichondrial layer was suggested as an intelligent functional composite for repair of craniofacial defects by Xu et al. The pseudoperichondrium was either placed in the center of the construct with the cell-fibrin mixture on both sides or on both surfaces with the cell-fibrin mixture in the middle.
Implantation of these constructs without pre-cultivation in vitro into the dorsal subcutaneous pocket of nude mice for 8 months resulted in good infiltration of the transplanted chondrocytes into the microporous structure of the polymer, the creation of a stable connection to the pseudoperichondrium and the development of an elastic construct for cartilage engineering [98, 99].
Fig. 4: Fibrin combined with a PGA non-woven mesh for cartilage engineering employing swine chondrocytes. Distributions of sulphated GAG, assessed with safranin-O staining within a composite cell construct after 7 days (left) and after 28 days (right) of in vitro culture. Dark segmented lines (left) are polymer fibers (with permission of Elsevier;
Ref: 43).
Cartilage is still the most important application of fibrin in tissue engineering. However, several publications complain about fast fibrin shrinkage and disintegration during chondrocyte cultivation. To overcome these problems, protease inhibitors or higher fibrinogen concentrations were employed as mentioned before [32, 36]. Other strategies include combinations with other hydrogels and polymeric scaffolds, respectively. Perka et al. mixed fibrin with stabilizing alginate, which can be easily removed prior to implantation [100]. Histology of human chondrocytes cultivated in this mixture for 30 days in vitro showed differentiated cells and formation of cartilaginous matrix. In an approach using a combination of fibrin and a polymeric scaffold, a high number of swine chondrocytes were suspended in fibrin gel and added to a PGA mesh [43]. It was reported that this combination already resulted in more mechanically stable constructs directly after cell seeding, i.e., the gel injected into the scaffold was more stable than the scaffold alone.
After 4 weeks of cultivation in vitro, the combination of fibrin and polymeric scaffold resulted in a higher amount of glycosaminoglycans, an effect that was partially attributed to increased matrix retention in the fibrin gel, and advanced mechanical stability of constructs for implantation compared to polymeric scaffold alone (Fig. 4).
Conclusion
We have summarized a wide range of fibrin applications in tissue engineering approaches to date. Fibrin glue can serve as a scaffold material alone, in combination with other hydrogels or porous polymeric scaffolds, and as delivery system for growth factors.
However, fibrin gels have a complex composition. Their appearance and mechanical strength varies enormously due to different components and concentrations. Therefore, it is extremely important to determine the exact matrix parameters necessary for a specific application. A soft gel is necessary for easy migration of cells inside the scaffold. The fast degradation of fibrin may be useful in sealants or for cell or growth factor delivery. In contrast, a strong and durable gel is essential for tissue engineering in vitro, where cells need enough time and sufficient mechanical integrity to produce their tissue-specific matrix. Also for an earlier implantation after cell-fibrin construct preparation, i.e. to shorten the in vitro culture period, mechanically stronger gels appear desirable. By varying the fibrin composition, cell number, and cultivation time, fibrin gels with different properties can be developed that are suitable for many different applications in clinical practice and the engineering of tissues. Such manipulations of fibrin properties may even eliminate the need for the more complex combinations with polymeric scaffolds or other hydrogels and supplements like protease inhibitors, in turn minimizing the risk of undesired side effects.
Acknowledgments
This work was supported by a grant from the Bavarian Research Foundation (‘Bayerische Forschungsstiftung’).
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Chapter 3
Factors Influencing Chondrocyte Behavior and Development of
Cartilaginous Tissue in Three-Dimensional Fibrin Gel
Daniela Eyrich 1, Hatem Sarhan 1, 2, Achim Goepferich 1, Torsten Blunk 1
1 Department of Pharmaceutical Technology, University of Regensburg, Universitaetsstrasse 31, 93051 Regensburg, Germany
2 Faculty of Pharmacy, El-minia Governate , El-minia University, El-minia, Egypt
Abstract
Despite several disadvantages, fibrin glue is a common material for three-dimensional cultivation of chondrocytes in the field of tissue engineering. Within this study, a modified long-term stable fibrin gel was tested for its general potential as scaffold in cartilage engineering using primary bovine chondrocytes in vitro. Cells suspended in 500 µl fibrin gels and cultured for 5 weeks in medium containing 5 % or 10 % FBS maintained their round and vital appearance and produced extracellular matrix containing GAG and collagen, though primarily concentrated in a small area around cells. In order to generate a uniform and coherent cartilaginous tissue, a lower fibrinogen concentration was found to improve homogenous distribution of extracellular matrix, however, at the same time reducing gel stability, compared to fibrin gels prepared with higher concentrations of fibrinogen. Furthermore, increasing cell density resulted in an increasingly coherent and homogenous extracellular matrix. However, when using a cell number of 40*106 or more per construct, matrix development decreased in the center of the gel, compared to the periphery of the constructs and to seeding a lower cell number. This effect was attributed to the large construct size resulting in insufficient diffusion and/or increased consumption of oxygen or nutrients. Moreover, a dynamic cultivation on an orbital shaker had enhancing effect neither on production of extracellular matrix components GAG and collagen nor on distribution of matrix, compared to statical cultivation. In contrast, addition of bioactive insulin to the culture medium containing 5 % FBS resulted in increased growth rate and development of extracellular matrix, even higher compared to cultivation with 10 % FBS in medium. However, this effect was still insufficient for the formation of a uniform and coherent cartilaginous tissue when employing 500 µl gels and a relatively low cell density.
On the other hand, the chondrocyte culture system can be used as a test system for delivery of growth factors or other bioactive molecules from controlled release devices. As an example, the model protein insulin slowly released from lipid microparticles enhanced production of cartilaginous extracellular matrix of cells suspended in fibrin gel and cultured in medium containing 5 % FBS. Taken together, these investigations clearly confirm the suitability of long-term stable fibrin gels for the use in cartilage tissue engineering, however, further investigations have to be conducted with regard to the generation of an adequate homogenous cartilaginous tissue.
Introduction
The aim of tissue engineering is to generate a new functional tissue by controlling the growth, differentiation and behavior of cell [1]. Besides the use of polymeric scaffolds, there has been enormous interest in using hydrogels as scaffold system [2-4]. Many hydrogels are similar to the extracellular matrix of various intact tissues, therefore, they are considered to be biocompatible and biodegradable. Hydrogels act as space filling agent and three-dimensional structure to organize the expanded cells, to maintain a specific shape and structural integrity, and to direct growth and formation for adequate new tissue development. They are suitable for many different cell types, as they are processed under relatively mild conditions, and may be easily delivered into the patient’s defect in a minimally invasive manner.
A common hydrogel in tissue engineering is fibrin [5]. Fibrin glue has been commonly used as a sealant and an adhesive in surgery, and has been commercially available for over 20 years in surgery and clinical practice [6-9]. In order for fibrin gels to be utilized as a tissue scaffold, the material must provide an environment enabling adequate cellular function, e.g. cell migration, proliferation, and differentiation, and must allow for tissue development. It has been shown that chondrocytes in fibrin gel retain their round and vital morphology, do not dedifferentiate, and produce extracellular matrix [10, 11]. The glue components fibrinogen and thrombin are thought to modulate the attachment, migration, and proliferation of different cell types, e.g. chondrocytes [12-16]. However, a fibrin characteristic is an increasing instability and solubility over time [17]. Commercial fibrin glues tend to shrink and disintegrate in vitro and in vivo after a few days and almost completely dissolve within 4 weeks, therefore they cannot be used in many applications in tissue engineering, where a shape-specific scaffold is preferred [10, 18-20]. Therefore, in modifying various fibrin parameters, our group has recently developed a transparent gel, that is stable in vitro for at least one year. Fibrin optimization as well as mechanical and rheological properties of the new gel will be discussed in detail in chapter 4.
Within this study, the newly developed fibrin gel was tested for its general potential for the use in cartilage tissue engineering. Therefore, primary bovine chondrocytes were suspended in fibrin, and cell behavior, cell morphology, as well as development of cartilaginous extracellular matrix were evaluated. With the objective to optimize chondrocyte culture, the effect of dynamical cultivation as well as of exogenous insulin on cartilaginous tissue development was investigated. Furthermore, in order to obtain a
uniform and coherent new tissue, influence of fibrinogen concentration as well as initially seeded cell number on cell behavior as well as matrix development and distribution were analyzed. Finally, the established chondrocyte culture system was tested for the use in evaluating controlled release of bioactive molecules from microparticles, using insulin as a potent cartilage-effective model drug. We tested the effect of insulin slowly released from incorporated lipid microparticles on chondrocytes grown within the long-term stable fibrin gels.
Materials and Methods
Materials
Aprotinin solution (Trasylol®) was bought from Bayer (Leverkusen, Germany). Thrombin (as a part of Tissucol®), thrombin dilution buffer and the commercially available fibrin glue kit Tissucol® was kindly provided by Baxter (Unterschleißheim, Germany). Bovine fibrinogen was purchased from Sigma-Aldrich (Taufkirchen, Germany).
Knee joints from three-months-old bovine calves were obtained from a local abattoir within 12-18 hours of slaughter. Type II collagenase and papainase were purchased from Worthington (CellSystem, St. Katharinen, Germany). Dulbecco’s Modified Eagle’s Medium (DMEM) with 4.5 g/l glucose, fetal bovine serum (FBS), MEM non-essential amino acid solution, penicillin, streptomycin, N-2-hydroxyethylpiperazine-N´-2- ethanesulfonic acid (HEPES buffer), phosphate buffer solution (PBS buffer) and trypsin EDTA were obtained from Gibco (Karlsruhe, Germany). 149 µm pore size polypropylene filters were purchased from Spectrum (Rancho Dominguez, CA, USA).
Ascorbic acid, deoxyribonucleic acid, diaminobenzidine, dimethylmethylene blue, glutaraldehyde, glycine, hematoxylin, proline and safranin-O were purchased from Sigma- Aldrich (Taufkirchen, Germany). Bovine insulin from bovine pancreas, chloramin-T, formalin 37%, and p-dimethylaminobenzaldehyde (p-DAB) were from Merck (Darmstadt, Germany). Hoechst 33258 dye was obtained from Polysciences (Warrington, PA, USA) and L-hydroxyproline from Fluka (Neu-Ulm, Germany). Chondroitin sulfate was from ICN (Aurora, Ohio, USA) and glycerol tripalmitate (Dynasan116®) was provided by Sasol AG (Witten, Germany).
Cell culture plastics were purchased from Corning Costar (Bodenheim, Germany).